Communication between T cells and antigen-presenting cells (APCs) via the immunological synapse (IS) forms an important regulatory point of the immune system [2, 3]. It is hypothesized that the spatial organization of receptor/ligand complexes within the IS is an important part of the language of T cell-APC communication but how this spacing influences cell signaling and the underlying mechanisms remain unclear. Moreover, certain subsets of cell types are rarely available from donors and the rarity of target T cells poses significant challenges in investigation of those cells. This proposed project seeks to first develop an innovative microfluidic platform allowing the efficient placement small numbers of T cells within a small, defined experimental zone and then be used to answer important questions on T cell-APC communication at IS. The overall approach includes: (1) creation of various artificial APC periodic pattern arrays by microcontact printing, (2) design and fabrication of microfluidic platform integrated to a variety of microcontact-printed APC in order to trap and precisely locate a small number of T cells on those patterns with the help of electromagnetic system, and (3) investigation of the selectively positioned T cells on those patterns by microscopic visualization and immunoassays. With the microfluidic platform we will develop as a first step of this proposal, we will test two specific hypotheses. The first hypothesis focuses on how the internal structure of the IS influences on T cell activation and signaling. We will determine whether the lateral organization of T cell receptor (TCR) and CD28 signaling in the IS can modulate IL-2 secretion by mouse CD4+ Tcells isolated from peripheral blood as similarly shown by mouse nave CD4+ T cells from lymph nodes [4]. The second hypothesis focuses on signaling within regulatory T cells (Tregs) that are important in suppressing the function of other T cells. PKC polarization in Tregs interacting with CD3 and ICAM-1 was reported very different than in effector T cells (Teffs) and blockade of PKC from proximal IS enhanced suppressive function of Tregs [5]. We will determine how PKC localization affects signaling within IL-17-producing T cells (Th17) in comparison to regulatory T cells (Tregs) because they share partly differentiation pathway but play fairly opposite functions. Successful completion of the proposed studies will provide important insights into the two complementary projects stated above. Longterm goal of the application is to provide peer immunologists with an innovative tool to investigate rarely available T cell subsets in more controlled condition and will help provide new insight into the communication between T cells and antigen presenting cells (APCs), thus advance treatment of immune disease such as rheumatoid arthritis. This platform also has wide application in other cellular systems, including investigation of cancer and stem cell physiologies.